Vaccines protect against a wide variety of infectious diseases. Many vaccines are produced by inactivated or attenuated pathogens which are injected into a subject, whereas others, so called ‘subunit vaccines’, are made from proteins or polysaccharides displayed on the surface of the pathogen. Subunit vaccines are preferred over inactivated or attenuated pathogens as they tend to cause fewer side effects. However, the development and production of a subunit vaccine requires the identification and isolation of protective antigens from the pathogenic organism, and moreover subunit vaccines based on polysaccharide antigens invoke often a T-cell independent immune response which results in low antibody titre, short half-life of the antibodies and low affinity for a specific antigen.
The development of subunit vaccines is an active research area and it has been recognized that the immunogenicity of polysaccharide antigens can be enhanced by conjugation to a protein carrier. Glycoconjugates have the ability to induce both humoral and adaptive immune responses. Currently licensed human glycoconjugate vaccines include those against Haemophilus influenzae, Neisserria meningitidis and Streptococcus pneumoniae, by which bacterial polysaccharides are chemically bound to carrier proteins. The H. influenzae type B (Hib) vaccine or Prevnar®, a 13-valent capsule-based glycoconjugate vaccine protective against diseases caused by S. pneumonia, employs the carrier protein iCRM197, a non-toxic version of diphtheria toxin isolated from Corynebacterium diphtheria. 
Although these vaccines are effective, their production requires both the purification of polysaccharide glycan from the native pathogen and the chemical coupling of the sugar to a suitable protein carrier, which can lead to impure products, low yields, variation between batches of conjugates and poses a biohazard as the handling of the pathogenic organism is unavoidable. This process is highly costly, inefficient and time consuming. The use of organic systems represents a more rapid and economical method for the production of glycoconjugates.
So far several pathogenic bacteria have been identified forming glycoproteins and the genes involved identified. The gram negative pathogenic bacterium Campylobacter jejuni harbours a gene cluster involved in the synthesis of lipo-oligosaccharides and N-linked glycoproteins. The protein glycosylation locus, a cluster of 12 genes comprising pgIA-pgIG, is involved in the glycosylation of over 30 glycoproteins. Part of the gene cluster is PgIB, an oligosaccharyltransferase catalysing the transfer of glycans on to a wide range of different non-species related protein acceptors, indicating broad substrate specificity. Moreover PgIB when expressed in E. coli has been used to produce novel glycoconjugates providing a genetic tool to express heterologous recombinant glycoproteins. Production of glycoconjugate vaccines in a bacterial system are disclosed in patent application WO2009/104074. Glycoconjugates comprising a protein carrier and an antigenic polysaccharide O-antigen form Shigella, E. coli and Pseudomonas aeruginosa using the oligosaccharyltransferase PgIB were produced, and bioconjugates against the Shigella O1 polysaccharide were shown to elicit an immune response.
Tularemia, also known as lemming or rabbit fever, is common in wild rodents and can be passed on to humans by contact with infected animal tissues, ticks or biting flies, or by inhalation of the infectious organism. Tularemia is found in North America, parts of Europe and Asia and is caused by the gram-negative coccobacillus Francisella tularensis. F. tularensis is highly infectious, with a mortality rate of up to 30% and based on the above listed characteristics classified as a Class A bioterrorism agent. Tularemia is difficult to diagnose, however, can be treated although with varying success with antibiotics. There are no vaccines available yet.
Recent studies suggest a protective effect using purified lipopolysaccharide (LPS) comprising an O-antigen from F. tularensis in a murine infection model; however, the development of glycoprotein vaccines protecting against highly infectious pathogens to high quantities using current methods are associated with high safety concerns.
We disclose a novel bacterial protein glycan coupling technology (PGCT) that allows the production of protective vaccines from the highly virulent wild-type strain of F. tularensis holarctica. The recombinant glycoconjugate was easily purified and was capable of providing significant protection against subsequent challenge when compared to LPS based vaccine treatments.